Abstract This proposal is focused on structural and ligand binding studies of Tau, one of the primary proteins involved in Alzheimer's disease. Our goals are to improve methods for imaging Tau in vivo, and to provide a high resolution structure to enable design of inhibitors of Tau aggregation. To accomplish these goals we will employ magic angle spinning (MAS NMR) to examine well ordered fibrils of Tau. Recently, our collaborator has expressed Tau296-351 in high yield, so that it aggregates reproducibly, indicating the formation of well ordered fibrils. The expression system will allow isotopic 13C/15N labeling to determine the atomic resolution structure with NMR. These samples will permit us to pursue the following 3 specific aims: 2.1 Aim 1: High resolution structures of Alzheimer's disease-related NFTs A cryo-EM study has identified two Tau fibril morphologies in samples from the brain of an Alzheimer's patient: paired helical filaments (PHF) and straight filaments (SF) with a core comprised of residues 306- 378 of Tau. However, the 3.4-3.5 resolution of the cryo-EM maps of Tau fibrils is not sufficient to acquire a complete atomic level structure. Thus, Aim 1 of this proposal is to use MAS NMR experiments, including DNP enhanced measurements, to refine the structure of Tau fibrils to atomic level resolution (? 1.5 ). We will produce a full set of chemical shift assignments for Tau which are required to accomplish Aim 2. 2.2 Aim 2: PET ligand binding sites and mechanisms Positron emission tomography (PET) scans using ligand molecules (radiotracers) containing radioactive isotopes that bind to Tau fibrils are an essential technique in the diagnosis of Alzheimer's disease. Since a high resolution structure is not available, there is little guidance for synthetic chemists to design radiotracers with high affinity binding. Our study will address these questions with structural studies of the binding of two radiotracers 18F-THK-5117 and 18F-T807, which are known to favor binding to Tau over A?. A knowledge of their binding mechanism will facilitate design of new agents of greated versatility. 2.3 Aim 3: 1H-detected DNP Our third goal is to combine two powerful MAS NMR techniques: 1H detection and dynamic nuclear polarization (DNP) to enable studies of tissue samples. Conventional MAS NMR experiments that use 3.2 mm rotor technology (spinning ~24 kHz) do not attenuate 1H homonuclear dipole couplings, resulting in broad peaks and poor resolution. Recently 0.7 mm MAS rotors capable of spinning at ~110 kHz, enable high-resolution 1H detection and require <0.5 mg of sample. Combining DNP and 0.7 mm rotors will permit studies of the binding of imaging agents Tau, A?, and other interesting to in vivo fibril samples.